System for Extended Storage of Red Blood Cells and Methods of Use

ABSTRACT

A system and methodology for the preservation of red blood cells is described in which red blood cells are oxygen or oxygen and carbon dioxide depleted, treated and are stored in an anaerobic environment to optimize preparation for transfusion. More particularly, a system and method for extended storage of red blood cells from collection to transfusion that optimizes red blood cells prior to transfusion is described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.Provisional application 61/504,640, filed on Jul. 5, 2011, and U.S.Provisional Application No. 61/504,644, filed Jul. 5, 2011; is a CIP ofU.S. patent application Ser. No. 12/901,350, filed on Oct. 8, 2010,which claims the benefit of U.S. Provisional Application No. 61/311,693filed on May 5, 2010; is a CIP of U.S. patent application Ser. No.13/115,532, filed May 25, 2011 which is a CON of U.S. patent ApplicationSer. No. 12/903,057, filed on Oct. 12, 2010 (abandoned), which claimsthe benefit of U.S. Provisional Application No. 61/250,661 filed on Oct.12, 200 9; and is a CIP of U.S. patent application Ser. No. 13/289,722,filed on Nov. 4, 2011, which claims the benefit of U.S. ProvisionalPatent Application Ser. No. 61/410,684 filed on Nov. 5, 2010, thecontents of each of which are incorporated herein by reference in theirentireties.

FIELD OF THE DISCLOSURE

The present disclosure relates to the systems and methods for thepreservation of blood and red blood cells. More particularly, thedisclosure relates to the systems and methods for the prolongedanaerobic storage of red blood cells from collection to transfusion.

BACKGROUND OF THE INVENTION

The supplies of liquid blood are currently limited by storage systemsused in conventional blood storage practice. Using current systems,stored blood expires after a period of about 42 days of refrigeratedstorage at a temperature above freezing (i.e., 4° C.) as packed bloodcell preparations. Expired blood cannot be used and must be discardedbecause it will harm the ultimate recipient. One of the primary reasonsfor blood spoilage is its continued metabolic activity after it isstored. For example, in 200 7, more than 45 million units of red bloodcells (RBCs) were collected and stored globally (15.6 million in theUS). During refrigerated storage, RBCs become progressively damaged bystorage lesions. When transfused within the current 6-week limit, storedRBCs have lower quality (fraction of RBC removed; compromised O₂delivery capacity) as well as potential toxicity, often manifested asside effects of transfusion therapy. These storage lesions are observedas altered biochemical and physical parameters associated with storedcells. Examples of these include in vitro measured parameters such asreduced metabolite levels (ATP and 2,3-DPG), reduced surface area,echinocytosis, phosphatidylserine exposure, and reduced deformability.

Stored blood undergoes steady deterioration which is partly caused byhemolysis, hemoglobin degradation and reduced adenosine triphosphate(ATP) concentration that occur during the storage period. These reasonsand others limit the amount of readily available high quality bloodneeded for transfusions.

As discussed above, when RBCs are stored under refrigeration attemperatures above freezing (e.g., 1-6° C., standard storage conditions)in a blood storage bag, away from mechanical stress and the constantlycycling environment of the circulation, the senescence process ispartially suspended. However, with the lack of constant nutrientreplenishment and waste removal under refrigerated storage, RBCs aregradually damaged, resulting in compromised physiological functions. Byway of example, the following problems occur during extended storage:

-   -   When RBCs are stored for an extended period, storage lesions        accumulate and deteriorate RBCs and cause up to 1% of RBCs to be        hemolyzed during storage and up to 25% to be removed shortly        after transfusion.    -   Non-viable RBCs cause iron overload in chronically transfused        patients.    -   Transfusion does not always achieve the intended outcome of        increased tissue perfusion.    -   Hemoglobin in RBCs do not release oxygen efficiently at tissues        due to loss of 2,3-DPG.    -   RBCs are not able to enter and perfuse capillary beds due to        loss of deformability.

Transfusing RBCs stored for longer periods may result in highermorbidity and longer hospital stays compared to transfusing “fresher”red cells. Higher morbidity and longer hospital stays result with RBCsthat are stored longer than 6 weeks, in comparison to fresher red cells.For example, negative clinical outcomes in cardiac surgery occur whenusing ‘older’ blood; multiple organ failure in surgical patientsreflecting the age of transfused red cells; correlation between olderunits and increased mortality in severe sepsis; failure to improve O₂utilization attributed to decreased 2,3-DPG and decreased cardiac indexassociated with increased blood viscosity

This evidence suggests that the ineffectiveness and negativeconsequences of transfusion is attributable at least in part to thecompromising effects of extended storage of RBCs. In addition toimmediate removal by the recipient of certain RBCs, consequences of RBCstorage lesions include: (i) Depletion of ATP (loss of RBC's ability todilate the pre-capillary arteriole); (ii) Depletion of 2,3-DPG; (iii)Accumulation of oxidative damage caused by reactive oxygen species (ROS)formed by the reaction of denatured hemoglobin with O₂; and (iv)Decreased RBC deformability and increased RBC viscosity-caused in partby oxidative damage to membrane and cytoskeleton. Less deformable RBCsare excluded from capillary channels resulting in low capillaryoccupancy and reduced tissue perfusion. Massive transfusion ofun-deformable cells may also contribute to multiple organ failure byblocking the organs' capillary beds. After transfusion, 2,3-DPG issynthesized relatively quickly in vivo to ˜50% of the normal level in aslittle as 7 hours and to ˜95% of the normal level in 2-3 days. However,since 2,3-DPG-depleted cells do not recover their levels immediately,O₂-carrying capacity is compromised to the detriment of critically illpatients requiring immediate O₂ delivery and tissue perfusion. There arenumerous reports that emphasize the importance of RBCs with high oxygencarrying capacity in such clinical situations.

Storage of frozen blood is known in the art but such frozen blood haslimitations. For a number of years, frozen blood has been used by bloodbanks and the military for certain high-demand and rare types of blood.However, frozen blood is difficult to handle. It must be thawed whichmakes it impractical for emergency situations. Once blood is thawed, itmust be used within 48 hours. U.S. Pat. No. 6,413,713 to Serebrennikovis directed to a method of storing blood at temperatures below 0° C.

U.S. Pat. No. 4,769,318 to Hamasaki et al. and U.S. Pat. No. 4,880,786to Sasakawa et al. are directed to additive solutions for bloodpreservation and activation. U.S. Pat. No. 5,624,794 to Bitensky et al.,U.S. Pat. No. 6,162,396 to Bitensky et al., and U.S. Pat. No. 5,476,764are directed to the storage of red blood cells under oxygen-depletedconditions. U.S. Pat. No. 5,789,151 to Bitensky et al. is directed toblood storage additive solutions.

Additive solutions for blood preservation and activation are known inthe art. For example, Rejuvesol (available from enCyte Corp., Braintree,Mass.) is added to blood after cold storage (i.e., 4° C.) just prior totransfusion or prior to freezing (i.e., at −80° C. with glycerol) forextended storage. U.S. Pat. No. 6,447,987 to Hess et al. is directed toadditive solutions for the refrigerated storage of human red bloodcells.

The effects of elevation and preservation of ATP levels in blood storagesituations has been studied. For example, in “Studies In Red Blood CellPreservation-7. In Vivo and in Vitro Studies With A ModifiedPhosphate-Ammonium Additive Solution,” by Greenwalt et al., Vox Sang 65,87-94 (1993), the authors determined that the experimental additivesolution (EAS-2) containing in mM: 20 NH₄Cl, 30 Na₂HPO₄, 2 adenine, 110dextrose, 55 mannitol, pH 7.15, is useful in extending the storageshelf-life of human RBCs from the current standard of 5-6 weeks to animproved standard of 8-9 weeks. Packed RBCs are suitable for transfusionfollowing the removal of the supernatant with a single washing step.Greenwalt et al. also conclude that factors other than ATP concentrationappear to play an increasingly important role in determining RBCviability after 50 days of storage. They cite the results of L. Wood andE. Beutler in “The Viability Of Human Blood Stored In Phosphate AdenineMedia,” Transfusion 7, 401-408 (1967), find in their own experimentsthat the relationship between ATP concentration and 24-hour RBC survivalmeasurements appear to become less clear after about 8 weeks of storage.E. Beutler and C. West restate that the relationship between red cellATP concentration and viability is a weak one after prolonged periods ofstorage in “Storage Of Red Cell Concentrates In CPD-A2 For 42 and 49Days,” J. Lab. Clin. Med. 102, 53-62 (1983).

In “Effects Of Oxygen On Red Cells During Liquid Storage at +4° C.,” byHogman et al., Vox Sang 51, 27-34 (1986), the authors discuss that redcell content of ATP is slightly better maintained in anaerobic chamberthan at ambient air storage after 2-3 weeks. Venous blood wasrefrigerated and deprived of additional oxygen during storage, byplacing the oxygen-permeable storage bags in a nitrogen environment andthereby gradually reducing the level of oxygen saturation. The reductionin oxygen concentration occurs slowly during storage at 4° C., and isfar from complete, starting at about 60% and reaching about 30%hemoglobin saturation at 5 weeks. No conclusion could be drawnconcerning the effects of this procedure on the overall quality ofstored cells. These authors did not address or significantly reduce theoxygen-dependent damage to hemoglobin and the oxygen-mediated damagecaused by hemoglobin breakdown products.

Many patents have addressed different aspects of blood storage. One suchpatent is U.S. Pat. No. 4,837,047 to Sato et al. which relates to acontainer for storing blood for a long period of time to keep thequality of the blood in good condition. Sato et al. is directed atimproving the storage life of the stored blood by maintaining a partialpressure of carbon dioxide gas in the blood at a low level. Such partialpressure is apparently obtained through normalization with the outsideatmosphere. The container is made of a synthetic resin film which has ahigh permeability to carbon dioxide gas for the purpose of making itpossible for the carbon dioxide gas to easily diffuse from the blood tooutside. However, the problems caused by the interaction of the oxygenand hemoglobin in the blood are not addressed.

Another patent, U.S. Pat. No. 5,529,821 to Ishikawa et al. relates to acontainer and a method for the storage of blood to prevent adhesion ofthe blood to the container. Blood is stored in containers composed of asheet material having a plurality of layers where a first sheet whichcontacts the blood substantially prevents the activation and adhesion ofblood platelets to the layer. Again, however, the problems caused by theinteraction of the oxygen and hemoglobin in the blood are not addressed.

In light of current technology, there is a need to improve the qualityof red blood cells that are to be stored and to extend the storage lifeof such red blood cells in advance of transfusion to minimize morbidityassociated with transfusions.

SUMMARY OF THE INVENTION

To address such needs and others, the present disclosure includes andprovides a system and methodology for the preservation of red bloodcells is provided in which red blood cells are, e.g., oxygen and carbondioxide depleted, undergo treatment and are stored in an anaerobicenvironment to optimize preparation for transfusion.

The present disclosure includes a system and method for extended storageof red blood cells from collection to transfusion that optimizes redblood cells prior to transfusion.

The present disclosure provides for, and includes, a method forpreparing red blood cells (RBCs) including obtaining whole blood,separating the RBCs from the whole blood to form packed RBCs, depletingoxygen to form oxygen depleted RBCs or depleting oxygen and carbondioxide to form oxygen and carbon dioxide depleted RBCs and storing theoxygen depleted or oxygen and carbon dioxide depleted RBCs in ananaerobic storage environment to maintain an oxygen depleted or oxygenand carbon dioxide depleted condition.

In aspects of the present disclosure, the method may further includeadding an additive solution to the packed RBCs to form a suspension. Insome aspects the additive solution may include AS-1, AS-3, AS-5, SAGM,PAGG-SM, PAGG-GM, MAP, SOLX, ESOL, EAS61, OFAS1 or OFAS3 alone or incombination. In a further aspect, the additive solution may have a pHfrom 5.0 to 9.0. In another aspect, the additive may include anantioxidant. In some aspects according the present disclosure, theantioxidant may be quercetin, alpha-tocopheral, ascorbic acid, or enzymeinhibitors for oxidases.

In aspects of the present disclosure, an integrated blood storage systemand method can include an oxygen and carbon dioxide removal system, ablood storage system and a pre-transfusion procedure to prepare thestored blood for transfusion.

Additionally, the present disclosure also includes a system and a methodthat may incorporate leukoreduction and editing steps to optimize RBCsin preparation for transfusion. Leukoreduction may include removingwhite blood cells that can carry viruses and cause fevers. Editing caninclude removing RBCs that exhibit indications of being compromised.

Accordingly, the present disclosure also provides a novel procedure forblood storage which addresses at least the problems of hemoglobindegradation, red blood cell lysis (hemolysis) and ATP and 2-3 DPGdepletion in a manner consistent with the practice of autologoustransfusion and enhanced heterologous transfusion logistics, and whichachieves significant prolongation of the time during which refrigeratedstorage of red blood cells is not detrimental to their subsequent use.

The present disclosure further provides for a system and methodology forenhancing the effect of irradiation and stabilizing red cells prior tostorage or during/after storage in preparation for transfusion.

The present disclosure further provides for a system and methodology forreducing the growth of aerobic bacteria and parasites present in redblood cells prior to storage or during storage in preparation fortransfusion.

The present disclosure further provides for a system and methodology forminimizing hemolysis and morphology changes of red blood cells duringstorage in non DEHP storage bags.

The present disclosure further provides for a system and methodology forstabilizing and enhancing pathogen inactivation of red blood cells priorto storage or during storage in preparation for transfusion.

The present disclosure, still further provides for a system andmethodology for providing nitric oxide to red blood cells duringstorage, after storage and immediately prior to transfusion, e.g., topermit vasodilation of the vessels of the recipient of the RBCs.

The present disclosure, still yet further provides for a system andmethodology for reducing the volume of red blood cells after storage andre-oxygenating such RBCs immediately prior to transfusion.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an exemplary flowchart of the components andmethodology from blood collection to transfusion using a blood anaerobicstorage system of the present disclosure;

FIG. 2 illustrates an exemplary system according to the FIG. 1 of thepresent disclosure in which, blood is collected, components areseparated, optional additive solution is added to packed RBC,leukoreduced then stored anaerobically;

FIGS. 3a and 3b illustrate the effects of, oxygen and oxygen and carbondioxide depletion on ATP and DPG, respectively, during extended storagein OFAS3 additive solution;

FIGS. 4a and 4b illustrate a partial detailed perspective view of an RBCinlet portion of the combination leukoreduction filter and O₂/CO₂depletion device according to the system of FIG. 5;

FIG. 5 illustrates a pre-storage oxygen, carbon dioxide oxygen andcarbon dioxide depletion device of the present disclosure;

FIG. 6 illustrates a cross-section view of the depletion device of thedevice of FIG. 5;

FIGS. 7A through 7D illustrate cross-section views of embodiments ofdepletion devices;

FIG. 8 illustrates the starting and ending partial pressures of oxygenand carbon dioxide, respectively, in red blood cells;

FIG. 9 illustrates the ending partial pressure of oxygen in RBCs as afunction of flow rate of RBCs and how depletion varies according to flowrate of RBCs when device similar to FIG. 5 was fed with RBC suspensionof 16.5 Torr;

FIG. 10 illustrates alternative oxygen/carbon dioxide depletion deviceincorporating leukoreduction and plasma separation components;

FIGS. 11a and 11b illustrate alternative blood bags according to thepresent disclosure;

FIGS. 12A through 12B illustrate embodiments of a blood storage bagaccording to the present disclosure;

FIG. 13 illustrates an alternative configuration according to thepresent disclosure;

FIG. 14 illustrates an aspect for volume reduction;

FIG. 15 illustrates a comparison of ATP of RBCs stored in accordancewith the present disclosure;

FIG. 16 illustrates a comparison of 2,3 DPG of RBCs stored in accordancewith the present disclosure;

FIG. 17 illustrates a comparison of hemolysis of RBCs stored inaccordance with the present disclosure;

FIG. 18 illustrates a transfusion kit having an oxygenation device tooxygenate RBCs in advance of transfusion;

FIG. 19 illustrates an alternative configuration according to thepresent disclosure, including leukoreduction, oxygen, carbon dioxide oroxygen and/or carbon dioxide depletion;

FIG. 20 illustrates an alternative configuration according to thepresent disclosure, including leukoreduction, oxygen, carbon dioxide oroxygen and/or carbon dioxide depletion and irradiation at differenttimes during aerobic and anaerobic conditions of RBCs;

FIG. 21 illustrates an alternative configuration according to thepresent disclosure, including leukoreduction, oxygen, carbon dioxide oroxygen and/or carbon dioxide depletion and re-oxygenation immediatelyprior to transfusion to a recipient;

FIG. 22 illustrates an alternative configuration according to thepresent disclosure, including leukoreduction, oxygen, carbon dioxide oroxygen and/or carbon dioxide depletion, and pathogen inactivation atvarious possible times during collection and storage; and

FIG. 23 illustrates an alternative configuration according to thepresent disclosure, including leukoreduction, oxygen, carbon dioxide oroxygen and/or carbon dioxide depletion, and nitric oxide addition atvarious possible times during storage.

DETAILED DESCRIPTION

The transfusion of red blood cells (RBCs) is a life-saving therapy aimedat improving oxygenation of the tissues and vital end organs in severelyanemic patients. The majority of RBC units used for transfusion arestored at 1-6° C. for up to 42 days in an oxygen-permeablepolyvinylchloride blood bag that contains additive/preservativesolution.

Exemplary Definitions:

Blood Donor: Whole blood is preferably donated from a healthy individualor donor and held in a blood bank for later use to be ultimately used bya recipient. Subjects who are scheduled for surgery or other treatmentmay donate blood for themselves in a process known as autologous blooddonation. Alternatively, blood is donated for use by another in aprocess known as heterologous transfusion. The collection of a wholeblood sample drawn from a donor, or in the case of an autologoustransfusion from a patient, may be accomplished by techniques known inthe art, such as through donation or apheresis.

Whole Blood: Whole blood is a suspension of blood cells that containsred blood cells, white blood cells, platelets suspended in plasma,including electrolytes, hormones, vitamins, antibodies, etc.

Red Blood Cells (RBCs): Human red blood cells in vivo are in a dynamicstate. In whole blood, white blood cells are normally present in therange between 4,300 and 10,800 cells/μL and the normal RBC range at sealevel is 5.4 million/μL (+ 0.8) for men and 4.8 million μL (+ 0.6) forwomen. The red blood cells contain hemoglobin, the iron-containingprotein that carries oxygen throughout the body and gives red blood itscolor. The percentage of blood volume composed of red blood cells iscalled the hematocrit. Packed red blood cells may be prepared from wholeblood using centrifugation techniques commonly known in the art. In anaspect according to the present disclosure, the packed red blood cellsmay be the blood component that is stored in the storage system forlater transfusion.

The normal life span of a red blood cell (RBC) is 120 days.Approximately 0.875% of the RBCs are retired every 24 hours by thespleen and new RBCs are made by the bone marrow. Consequently, whenblood is drawn from a donor, there are a percentage of white blood cellsand a spectrum of cells of different ages.

The main function of RBCs is to exchange oxygen and carbon dioxide atlung and tissues, and unlike other cells in body, it does not rely onoxygen in oxidative phosphorylation but entirely on glycolysis for ATPproduction. ATP is critical for viability of RBC and together with2,3-diphosphoglycerate (2,3-DPG), their free cytosolic concentrationsare tightly regulated by their function on feedback inhibition to keyenzymes in glycolytic pathway. Under a refrigerated storage condition,dis-inhibition of glycolytic pathway is desirable to overcome thegradual depletion of ATP and 2,3-DPG over several weeks of storage.Hemoglobin concentration in RBC is similar to 2,3-DPG and ATP, and itsdeoxygenated state has a binding pocked with high affinities for 2,3-DPGand ATP compared to oxy-hemoglobin. Thus, stripping this oxygen to few %occupancy (˜60% occupied when collected and processed) will cause uptakeof 2,3-DPG and ATP, resulting in reduced concentration of freemolecules, stimulating glycolytic flux.

Platelets: The platelets are small cellular components of blood thatfacilitate the clotting process by sticking to the lining of the bloodvessels. The platelets like the red blood cells are made by the bonemarrow and survive in the circulatory system for 9 to 10 days beforethey are removed by the spleen. Platelets are typically prepared using acentrifuge to separate the platelets from the plasma.

Plasma: Plasma is a protein-salt solution and the liquid portion of theblood in which red and white blood cells and platelets are suspended.Plasma is 90% water and constitutes about 55 percent of the bloodvolume. One of the primary functions of plasma is to assist in bloodclotting and immunity. Plasma is obtained by separating the liquidportion of the blood from the cells. Typically, plasma is separated fromthe cells by centrifugation. Centrifugation is the process used toseparate the components of the whole blood into the plasma, the whiteblood cells, the platelets and the packed red blood cells. Duringcentrifugation, the plasma will initially migrate to the top of a vesselduring a light spin. The plasma is then removed from the vessel. Thewhite blood cells and platelets are removed during a secondcentrifugation cycle to produce the packed red blood cells. Thisapplication will discuss an efficient alternative to using a centrifugethat minimizes the cost of traditionally used instrumentation.

In its most general form, the present disclosure provides for, andincludes, an integrated system and method for the preparation andextended storage of red blood cells, from receipt of whole blood from adonor until transfusion to a recipient. By way of example, FIG. 1illustrates an exemplary flowchart of the components and methodologyfrom blood collection from a blood donor 15 to transfusion to arecipient 50 using a anaerobic storage method 10 and system 20 throughPre-Storage Phase A, Storage Phase B in an anaerobic environment, andPost-Storage Phase C. However, as understood with reference to thepresent disclosure, various combinations of the disclosed systems andmethods are envisioned as within the scope of the disclosure, and theillustrated components and methodologies may be optionally substituted,eliminated or reordered.

By way of illustration, method 10 describes a storage system 20 thatincludes an optional additive addition, and oxygen, carbon dioxide, oroxygen and carbon dioxide (collectively referred to herein as O/CD)depletion of RBCs before and during storage, together with enhancingtreatments include leukoreduction, editing, pathogen reduction,irradiation and nitric oxide treatment and oxygen addition to enhancethe quality of stored RBCs and to optimize the transfusion process to arecipient and reduce morbidity associated with such transfusion.

Again referring to the drawings, and particular to FIG. 1, a method 10describes storage system 20 from collection from a donor 15 totransfusion to a recipient 50. System 20 shows a method that has threephases during which different sub-processes or steps may occur. Thethree phases are generally: Pre-Storage Phase A, Storage Phase B andPost-Storage Phase C. As shown in FIG. 1, different steps of the bloodstorage process 20 can occur at different phases to achieve optimalblood transfusion results. For example, irradiation can optionally occurduring Pre-Storage Phase A before oxygen removal, during Storage PhaseB, during the Post-Storage Phase C, during Storage Phase B and a portionof Pre-Storage Phase A and Post-Storage Phase C, or combinationsthereof, etc. Similarly, editing of RBCs (e.g., to remove moribund RBCs)can occur during Pre-storage Phase A, during Post-storage Phase C, or acombination thereof, etc. The anaerobic environment has synergisticrelationships with steps such as the addition of nitric oxide,irradiation and pathogen inactivation, that provide advantages to theRBCs that must occur in such anaerobic environment, as will be discussedbelow. Accordingly, there exist several different sequences for theblood storage processing according to the present disclosure.

Pre-storage Phase A, includes the time from collection from a donor tostorage in an anaerobic environment. During Phase A, whole blood may becollected from a donor, and the blood components, namely, plasma,platelets and RBCs may be separated. An optional additive solution maybe added to the whole blood to aid in storage and/or processing, asfurther described herein. Processing such as pathogen inactivation,leukoreduction and editing may occur during Pre-storage Phase A. DuringPhase A, oxygen, carbon dioxide, or oxygen and carbon dioxide (O/CD) aredepleted prior to Storage Phase B. O/CD may be depleted either by anoxygen, or oxygen and carbon dioxide depletion device (OCDD).

Storage Phase B is an anaerobic storage period, wherein RBCs are storedin an anaerobic storage environment.

Post-Storage Phase C, after storage in an anaerobic storage environmentbut prior to transfusion to recipient. Post-Storage Phase C may includeprocessing such as volume reduction, editing, cleansing during bufferexchange, the addition of either or both nitric oxide and oxygen, etc.

Referring to the drawings and in particular to FIG. 2, an exemplaryanaerobic storage system is shown and referenced using reference numeral25. In certain embodiments, system 25 may be constructed so as to bedisposable. Again, system 25 is an exemplary system, accordingly,different sub-processes or steps can occur at different times or duringdifferent phases as discussed above. Blood storage system 25 includes anoxygen/carbon dioxide depletion device 100 (OCDD 100), an anaerobicblood storage bag 200 and an optional additive solution bag 250.Components conventionally associated with the process of bloodcollection are a phlebotomy needle 16, a blood collection bag 35containing an anti-coagulant and a bag 45 containing plasma. Tubing canconnect the various components of the blood storage system 25 in variousconfigurations (one embodiment shown). OCDD 100 removes oxygen andcarbon dioxide from red blood cells traveling therethrough. System 25may also contains a leukoreduction filter 400, and editing device 500,an irradiation device 600, a pathogen inactivation device 700, a volumereduction device 800 and a nitric oxide device 900 to immediately supplynitric oxide to the RBCs in advance of transfusion to a recipient 50.System 25 can contain all or a combination of such devices 400 through900 in varying configurations as discussed below.

Components of system 25 are connected in a convention fashion. Tube 440connects collection bag 35 with leukoreduction filter 400. Tube 441connects solution bag 250 with collection bag 35. Tube 442 connectsplasma bag 45 with collection bag 35. Tube 443 connects leukoreductionfilter 400 with OCDD 100. Tube 444 connects OCDD 100 with blood storagebag 200. Blood storage system 25 is preferably a single-use, disposable,low cost system.

System components, namely, leukoreduction filter 400, editing device500, irradiation device 600, pathogen inactivation device 700, volumereduction device 800 and nitric oxide device 900, perform varioustherapies for the RBCs prior to transfusion. Depending upon thetherapies, such therapies are preferably performed on RBCs beforepassage through OCDD or after storage in storage bag 200. After beingdepleted in O/CD, RBCs are maintained in an oxygen, carbon dioxide, oroxygen and carbon dioxide depleted environment to ensure the desiredresults for the patient and to avoid morbidity commonly associated withtransfusions using stored RBCs.

In certain aspects, if desired, after packed RBCs are collected fromwhole blood obtained from donor 15, an optional additive solution, e.g.,from bag 250 may be provided to the packed RBCs to form a suspension ofpacked RBCs. Additive solutions may generally help to prevent rapiddeterioration of RBCs. Additive solution bag 250 may include an additivesolution optimized for anaerobic storage. For each of the severalembodiments addressed herein, an additive solution from bag 250 may beprovided prior to depleting O/CD from the RBCs. By way of example,between 50-300 ml of additive solution/unit of packed RBCs (450-500 mlwhole blood draw) may be added. In certain aspects, 100 to 110 ml ofadditive solution per unit of packed RBCs may be added. In anotheraspect, 50 to 100 ml of additive solution per unit of packed RBCs may beadded. In an aspect according the present disclosure, the 75 to 125 mlof additive solution per unit of packed RBCs may be added. In yetanother aspect according the present disclosure, the 90 to 120 ml ofadditive solution per unit of packed RBCs may be added.

By way of example, the additive solution may include an aqueous solutionof adenine, dextrose, mannitol, citrate ion, and dihydrogen phosphateion. Alternatively, additive solutions may include AS-1, AS-3, AS-5,SAGM, PAGG-SM, PAGG-GM, EAS61, OFAS1, OFAS3, MAP, ESOL, SOLX and anycombinations thereof. (See, Rossi's Principles of Transfusion Medicine4^(th) edition, Simon, T; Snyder, E, et al. Wiley-Blackwell; M Shimizu,H Fujii, H Mizoguchi, M Masuda, K Toyama, Rinsho Ketsueki et al.,“Multicenter clinical evaluation of red cell concentrates stored up to 6weeks in MAP, a new additive solution,” The Japanese Journal 33:148(1992); Dumont L J, Yoshida T, AuBuchon J P, “Anaerobic storage of redblood cells in a novel additive solution improves in vivo recovery,”Transfusion 49:458-64 (200 9); U.S. Pat. No. 5,789,151, issued Aug. 4,1998, entitled “Prolonged cold storage of red blood cells by oxygenremoval and additive usage,” issued Aug. 4, 1998; U.S. Pat. No.4,769,318 issued Sep. 6, 1988 to Hamasaki et al. entitled “AdditiveSolution for Blood Preservation and Activation; and U.S. Pat. No.6,162,396 to Bitensky et al. issued Dec. 19, 200 0 entitled “BloodStorage Device and Method for Oxygen Removal”; each of which are herebyincorporated by reference in their entireties).

Additive solution OFAS3 includes adenine, dextrose, mannitol, NaH₂PO₄,and optionally NaCl and/or NH₄Cl. Additive solution OFAS3 preferablyincludes ingredients having the following ranges: about 0.5-4.0mmole/liter of adenine, about 50-150 mmole/liter of dextrose, about20-70 mmole/liter of mannitol, about 0-100 mmole/liter of NaCl, about2-20 mmole/liter of NaH₂PO₄, and about 0-30 mmole/liter NH₄Cl.Preferably OFAS3 has an adjusted pH from about 5.5-7.5 and includesabout 2 mmole/liter adenine, about 110 mmole/liter dextrose, about 55mmole/liter NaCl, and about 12 mmole/liter NaH₂PO₄ and an adjusted pH ofabout 6.5. Additional embodiments of OFAS3 are provided in U.S. Pat. No.8,071,282, issued Dec. 6, 2011, which is herein incorporated byreference in its entirety.

TABLE 1 Ingredient Range (mM) Adenine 0.5-4.0  Dextrose 50-150 Mannitol0-70 NaCl  0-100 NaH₂PO₄ 2-20 NH₄Cl 0-30 Effective Osm 100-300  AdjustedpH 5.0-7.7  mL added 100-300 

OFAS3 has shown enhanced ATP levels and good in vivo recovery asdisclosed herein. FIG. 3a shows the effects of oxygen and oxygen andcarbon dioxide depletion on ATP during extended storage in oxygendepleted or anaerobic OFAS3 additive solution. FIG. 3b shows the effectsoxygen and oxygen and carbon dioxide depletion on 2, 3 DPG duringextended storage in oxygen depleted or anaerobic OFAS3 additivesolution. The highest ranges are from 8 to 30 days for ATP and from 0 to20 days for 2, 3 DPG. Ideally, RBCs would be transfused to recipient 50during such length of time.

To increase the time of acceptable in vivo recovery of RBCs in liquidstorage, attempts have been made to improve additive solutions andstorage processes. In “Studies In Red Blood Cell Preservation-7. In vivoand in vitro Studies With A Modified Phosphate-Ammonium AdditiveSolution,” by Greenwalt et al., Vox. Sang. 65:87-94 (1993), the authorsdetermined that the experimental additive solution (EAS-2) containing inmM: 20 NH₄Cl, 30 Na₂HPO₄, 2 adenine, 110 dextrose, 55 mannitol, pH 7.15,is useful in extending the storage shelf-life of human RBCs from thecurrent standard of 5-6 weeks to an improved standard of 8-9 weeks.However, packed RBCs stored in the medium were not directly infusiblebut required the removal of the supernatant with a washing step prior totransfusion due to the presence of ammonium in the additive solution.

In other embodiments, the additive solution may include antioxidants.Particularly preferred are antioxidants that may be active under minimaloxygen conditions and therefore, potentially synergistic in their actionin an anaerobic storage environment. For instance, the antioxidant maybe selected from quercetin and other bioflavonoids, alpha-tocopheral,ascorbic acid, ebselen, oxypurinol, hydrocortisone and other enzyme(oxidase) inhibitor molecules,and combinations thereof. Quercetin, aflavonoid with antioxidant activity is safe and efficient to act as anantioxidant when administered clinically. Quercetin scavenges oxygenradicals, inhibits lipid peroxidation in vitro, and has been shown toreduce erythrocyte membrane damage. In certain embodiments, theantioxidant may be a flavonol. In other aspects, the flavonoid may berutin or epicatechin. Ascorbic acid is very effective anti-oxidant, butcan also function as pro-oxidant. However, since relatively highconcentrations (˜10 mM, concentration necessary to be effective instored blood) and presence of iron (free or heme) and oxygen arenecessary for its pro-oxidant activity, the anaerobic conditions of thepresent disclosure should provide a low effective concentration of usewithout concern for an pro-oxidant activity.

Leukoreduction

As shown in FIG. 1, the whole blood, packed RBCs, or suspension of RBCsmay undergo leukoreduction 400. Leukoreduction is the general process ofremoving white blood cells from whole blood or red blood cells. Asshown, the leukoreduction may occur prior to or after depleting oxygen,carbon dioxide or oxygen and carbon dioxide (O/CD) to form O/CD depletedRBCs, and before, during or after storing in said anaerobic storageenvironment.

In accordance with certain aspects, referring to FIGS. 4a and 4b , acombination leukoreduction filter and OCDD 400 is shown. Combinationleukoreduction and OCDD filter 400 includes an inlet flow distributor410, a leukoreduction media 420, a plurality of hollow fibers and/orgas-permeable films or fibers 430, and a fiber/film support 440 to holdthe plurality of fibers and/or gas-permeable films or fibers 430.Plurality of hollow fibers and/or gas-permeable films or fibers 430 arefor the purpose of removing oxygen and or carbon dioxide from red bloodcells and will be discussed further below in conjunction with OCDD 101.

Leukoreduction media 420 is preferably a fibrous or a felt-likefiltering material that captures leukocytes (e.g., Pall Corporation),prior to such leukocytes travelling through plurality of hollow fibersand/or gas-permeable films or fibers 430 for oxygen, carbon dioxide, oroxygen and carbon dioxide depletion. In some aspects according to thepresent disclosure, the leukoreduction media may be a LeukoGuard-6® typefilter media. In an aspect the leukoreduction media 420 may beLeukotrap® Affinity Plus Prion and Leukocyte Reduction Filter media. Inan aspect the leukoreduction media 420 may be Leukotrap® media.Leukoreduction media 420 may comprise a fibrous medium, for example amedium prepared from melt-blown fibers, as disclosed in, for example,U.S. Pat. Nos. 4,880,548; 4,925,572, 5,152,905, 5,443,743, 6,231,770,and 7,361,277, each which are incorporated by reference in theirentireties. Each of the media, which can be preformed media, can includea plurality of layers, as disclosed in the U.S. Patents listed above.Fiber/film support 440 supports the plurality of fibers/films 430 in avertical configuration and may be made from a material such aspolyurethane or a similar material. Either whole blood or RBCs flowthrough media 420 leukoreduction process.

Method 10 shows that leukoreduction filter 400 or the process ofleukoreducing can optionally occur at whole blood stage or after RBCshave been separated from the plasma and platelets, before oxygen andcarbon dioxide have been removed or after oxygen and/or carbon dioxidedepletion in OCDD. In either case, leukoreduction can occur beforestorage of RBCs in a blood storage bag 200.

The benefits of leukoreduction are several. Leukoreduction may reducethe likelihood of fever in recipients, enhance RBC storagecharacteristics and reduce the transmission of viruses contained inleukocytes. Leukocytes in blood products can cause immunosuppressiveeffects and can pre-dispose recipients to an increased risk ofinfections, and may serve as vehicles of pathogen transmissions.Leukoreduction may reduce RBC storage lesions, reduce primaryalloimunization and reduce the total number of transfusion reactions ina recipient. By removing leukocytes from the RBCs before storage instorage bag 200, the deleterious effects of leukocytes highlighted abovecan be avoided and the quality of stored RBCs may be thereby increasedor enhanced.

Oxygen/Carbon Dioxide Removal

In some aspects according to the present disclosure, RBCs may be treatedto remove oxygen, carbon dioxide or oxygen and carbon dioxide in OCDD101, as shown in FIGS. 2, 5 and 6. OCDD 101 may have a housing 104, aninlet port 102 and an exit port 103, through which RBCs enter and exitOCDD 101, respectively. OCDD 101 of FIG. 6 represents one embodiment ofOCDD and contains an oxygen sorbent 110 at core 109. OCDD 101 mayalternatively contain a carbon dioxide sorbent or a combination ofoxygen and carbon dioxide sorbent. OCDD 101 may be comprised of adisposable housing having a series of hollow fibers and/or gas-permeablefilms or fibers 115 (or membranes) that are oxygen, carbon dioxide, oroxygen and carbon dioxide permeable. Optionally, housing 104 also has avent 114 for air to enter when draining the device after completing thedepletion process to allow maximal RBC recovery.

O/CD sorbent 110 may be a mixture of non-toxic inorganic and/or organicsalts and ferrous iron or other materials with high reactivity towardoxygen, carbon dioxide, or oxygen and carbon dioxide. O/CD sorbent 110may be made from particles that have significant reaction capacity forO₂ (e.g., more than 5 ml O₂/g) and can maintain the inside of OCDD 101less than, e.g., 0.01% which corresponds to pO₂ less than 0.08 mmHg.O/CD sorbent 110 may be either free or contained in an oxygen permeableenclosure, container, envelope, etc. For example, oxygen scavengers andcarbon dioxide scavengers are provided by Multisorb Technologies(Buffalo, N.Y.), or MGC (New York, N.Y.). Oxygen sorbents may exhibit asecondary functionality of carbon dioxide scavenging. Carbon dioxidescavengers include metal oxides and metal hydroxides. Metal oxides reactwith water to produce metal hydroxides. The metal hydroxide reacts withcarbon dioxide to form water and a metal carbonate. Such materials canbe blended to a desired ratio to achieve desired results.

In certain aspects, OCDD 101 of the present disclosure is configured tothroughput and deplete approximately 100 mL of oxygen from a unit ofblood. Alternatively, after passage of RBCs through OCDD, the oxygensaturation levels are reduced to less than 3 Torr in the RBCs.Alternatively, carbon dioxide levels are depleted in the RBCs to levelsof approximately 10 Torr.

Again referring to FIGS. 5 and 6, hollow fibers and/or gas-permeablefilms or fibers 115 may be constructed as membranes in a flat sheetform. Hollow fibers and/or gas-permeable films or fibers 115 may benon-porous materials that are capable of high O/CD permeability rates(polyolefins, silicones, epoxies, polyesters etc) and membrane arehydrophobic porous structures. These may be constructed of polymers(polyolefins, Teflon, PVDF, polysulfone) or inorganic materials(ceramics).

Referring to FIGS. 7B through 7D, alternative OCDD configurations (crosssectional views) are shown, with alternating sorbent 110 with hollowfibers 115 (embodiment of FIG. 6 is represented in FIG. 7A). In theembodiment of FIG. 6 and FIG. 7A, the characteristic diffusion time ofoxygen is approximately 7.5 seconds. The placement of sorbent materialrelative to hollow fibers is critical because the diffusion time isproportional to the inverse square of distance from sorbent to thefiber. If the distance between the sorbent and fiber is reduced by onehalf the diffusion time is reduced by one quarter.

TABLE 2 Example Specification Eternal Gas Pathways Eternal Gas PathwaysExample Serial #: Device 70 Fiber Type: Celgard Celgard 200/150-66FPI200/150-66FPI Number of Fibers/unit: 5000 5000 Active Length of 13 28Fibers (cm): Fiber OD (microns): 200 200 Fiber ID (microns): 150 150Total Length of Fibers 15 30 Active Fiber Surface 0.4084 0.8796 Area(m²):

In the oxygen/carbon dioxide depletion devices disclosed herein, aplurality of gas permeable films/membranes may be substituted for theplurality of hollow fibers/films. The films and fibers may be packed inany suitable configuration within the cartridge, such as linear orlongitudinal, spiral, or coil, so long as they can receive and conveyred blood cells.

The lowest oxygen saturation is achieved using devices in which thesorbent is placed close to fibers to enable rapid diffusion time.Additional factors that increase oxygen and/or carbon dioxide diffusionare larger active surface area of fibers exposed to sorbent materials.

Referring to FIG. 5 and FIG. 8, the graph shows the effect of OCDD 101on the partial pressure of RBCs that pass therethrough. At point A,prior to entry in OCDD 101, the partial pressure of oxygen in the RBCsis 16.8 Torr and at point B, the partial pressure of oxygen in the afteroxygen and carbon dioxide depletion device is approximately 3 Torr. Thepartial pressure of carbon dioxide is approximately 20 Torr at point Aand the partial pressure after RBCs pass through OCDD is approximately 3Torr.

FIG. 9 shows the partial pressure of oxygen in RBCs as a function ofmass flow rate of the RBCs through OCDD 101. The partial pressure ofoxygen in the gas surrounding hollow fiber as measured from the ventport 114 ranges from 1 to 0.5 Torr depending on the flow rate of theRBCs therethrough. FIG. 8 reveals the oxygen depletion that is providedby OCDD 101. The oxygen sensor at the outlet is enclosed in a bagflushed with nitrogen gas to increase the sensitivity of the pO₂measurement of blood; pO₂ surrounding the sensor is shown as ‘ambientair’ in FIG. 9.

The OCDD functions to deplete oxygen from RBCs from oxy-hemoglobinbecause more than 99% of such oxygen is hemoglobin-bound in venousblood. Preferably, the degree of oxygen saturation is to be reduced toless than 3 Torr within 48 hours of blood collection. The oxygendepletion is preferably accomplished at room temperature. The affinityof oxygen to hemoglobin is highly dependent on the temperature, with apartial pressure of 26 Torr at 37° C. dropping to ˜4 Torr at 4° C.Furthermore, this increase in O₂ affinity (K_(a) hemoglobin-oxygenbinding constant) is mainly due to reduction in O₂ release rate(k_(off)), resulting in an impractically low rate of oxygen removal onceRBC is cooled to 4° C. Thus, it places a constraint on oxygen strippingsuch that it may be preferable to accomplish it before RBC are cooled tostorage temperatures of 1° C. to 6° C.

As an alternative to or in addition to oxygen depletion, carbon dioxidedepletion has the beneficial effect of elevating DPG levels in red bloodcells. Carbon dioxide exists inside RBCs and in plasma in equilibriumwith HCO₃ ⁻ ion (carbonic acid). Carbon dioxide is mainly dissolved inRBC/plasma mixture as carbonic acid and rapid equilibrium between CO₂and carbonic acid is maintained by carbonic anhydrase inside RBC. Carbondioxide is freely permeable through RBC membrane, while HCO₃ ⁻ insideRBC and plasma is rapidly equilibrated by anion exchanger (band 3)protein. When CO₂ is removed from RBC suspension, it results in theknown alkalization of RBC interior and suspending medium. This resultsfrom removal of HCO₃ ⁻ inside and outside RBC; cytosolic HCO₃ ⁻ isconverted to CO₂ by carbonic anhydrase and removed, while plasma HCO₃ ⁻is removed via anion exchange inside RBC. Higher pH inside RBC is knownto enhance the rate of glycolysis and thereby increasing ATP and DPGlevels. ATP levels are higher in Ar/CO₂ (p<0.0001). DPG was maintainedbeyond 2 weeks in the Argon purged arm only (p<0.0001). Enhancedglycolysis rate is also predicted by dis-inhibition of key glycolyticenzymes via metabolic modulation and sequesterization of cytosolic-freeDPG upon deoxygenation of hemoglobin as a result of anaerobic condition.DPG was lost at the same rate in both control and Ar/CO₂ arms (p=0.6)despite thorough deoxygenation of hemoglobin, while very high levels ofATP were achieved with OFAS3 additive.

By depleting carbon dioxide in the OCDD, the pH of RBCs in cytosol isincreased. Further, 2, 3-DPG levels are increased for the first 3 weeksof storage and ATP level is maintained at high levels. These factorsenhance the viability of RBCs prior to being stored at oxygen depletedstorage in Phase B.

Referring to FIG. 10, a further embodiment of OCDD device 750 is shown,in which flow of oxygen free gas or oxygen free gas with carbon dioxidethrough the body of the device (two ports protruding on the left side ofcylinder) can be used to combination with a leukoreduction filter 710,OCDD 720, a plasma separator 730. Multifunction OCDD 750 eliminates theneed for centrifugation of the whole blood, received from donor 15,which is currently a necessity by using a centrifuge. By combining thesethree devices into a single device, the need for a separate centrifuge,a highly costly and cumbersome device, is eliminated. Plasma flowsthrough port 740 to a further collection bag for further processing.Accordingly, in this embodiment, whole blood can be collected from adonor, leukocytes can be removed, oxygen and or carbon dioxide can beremoved and plasma and platelets can be removed to pass RBCs throughdevice. The RBCs are then deposited into collection bag 200 afteradditive solution is added through the device for storage or transfusionto a recipient. Multifunction OCDD 750 as part of collection system 10and system 100 permit rapid transformation of whole blood to stored RBCsfor immediate storage or transfusion to a recipient.

Editing

Before oxygen and/or carbon dioxide are removed from the RBCs, the wholeblood or RBCs may be edited. Editing RBCs is the process of identifyingand removing blood cells that have a poor likelihood of surviving thetransfusion process or will likely die shortly after transfusion.Editing moribund RBCs, or dead or dying red blood cells, may be employedby using, for example, a filter-like editing device 500. Editing canoccur at various times during the storage process, e.g., as shown inFIG. 1. For example, editing can occur with whole blood or RBCs, beforebeing O/CD depleted and prior to storage in an anaerobic storageenvironment. While FIG. 1 shows the editing step being performed byediting device before oxygen and or carbon dioxide have been removed,editing can alternatively be performed at different stages of thestorage process. For example, editing can be performed immediatelybefore transfusion after storage in storage bag 200.

Editing can be important because a leading cause of morbidity andmortality to transfused patients is the non-viable portion of the bloodthat is transfused independent of any pathogen transmission. RBCs thatare compromised or that will be removed by the spleen by thereticuloendothelial system shortly after transfusion may threaten tooverwhelm the already compromised recipient. Up to 25% of transfusedcells are removed by recipient in the first twenty four hours aftertransfusion. These removed cells are harmful because they contributeimmediately to the excess iron burden of the recipient, which may be acritical parameter for chronically or massively transfused patients.Also, these cells may cause capillary blockage due to reduceddeformability or aggregate formation, leading to poor tissue perfusionand even organ failure. Thus, substantial benefits are expected if onecan remove these less viable RBCs prior to transfusion.

There are several techniques that may be used to edit the red bloodcells. The first technique is a centrifugation process to separate oldand young RBCs before storage based on characteristic buoyancies ofyoung and old RBCs.

A second technique applies a biomechanical stress, such as an osmoticshock, to hemolyze weak cells before or after storage in combinationwith a buffer exchange step. The applied biomechanical stressimmediately identifies those cells that are weak to rapidly contrastwith the stronger RBCS to enable mechanical separation. The weak RBCsare those that contribute to recipient morbidity and mortality,particularly, with individuals with already compromised or overloadedimmune systems. Up to 25% of RBCs that arrive to a recipient are alreadydead and can have deleterious effects on the recipient. By editing theRBCs, that number can be reduce by 50% to 75%.

A third technique applies to the deformability of the RBCs. Bump arraymicrofluidic devices containing staggered pillars (Huang, L. R., et al.,“Continuous particle separation through deterministic lateraldisplacement,”. Science 304(5673): 987-90 (200 4) herein incorporated byreference in its entirety), allow deformable RBCs to pass through thepillars while deformable RBCs can not pass through the pillars and arebumped into separate channels.

A further technique for editing the RBCs uses a filter system to removeRBC exhibiting a specific surface marker. RBCs exhibiting known surfacemarkers such as phosphatidiyserine or aggregated protein 3 can betrapped by a filter surface modified with high affinity ligand (e.g.,Annexin IV or antibodies against specific surface marker protein).

An additional technique uses the same high affinity ligands in thesecond technique, conjugated to make a multimeric molecule such thatRBCs exhibiting target surface markers forms aggregate. This can thenseparated by filtration or centrifugation.

Irradiation

A further processing step that RBCs may be subjected to is irradiation,e.g., by either gamma or X-ray radiation. Irradiation of RBCs is ofsignificance to avoid transfusion related complications.Transfusion-associated graft-versus-host disease (TA-GVHD) is a rare butnearly always fatal complication associated with transfusion therapy inseverely immuno-compromised blood recipients (for example, bone marrowtransplant recipient, patients receiving aggressive chemotherapy,premature neonates). Prevention of TA-GVHD requires complete removal of,or cessation of the proliferative potential of T-lymphocytes from donorblood. For instance, leukoreduction filters are not adequate inprevention of TA-GVHD because leukoreduction filters may not completelyeliminate lymphocytes. Thus, lymphocyte inactivation byX-/gamma-irradiation may be preferred for TA-GVHD prevention.

Gamma-irradiation abrogates proliferation of T-lymphocytes by damagingthe DNA directly and via reactive oxygen species (ROS), namely hydroxylradicals produced during gamma-radiolysis of water. Although RBCs do notcontain DNA, ROS generated by gamma-irradiation have been shown to causesignificant damage to the RBCs. The major damage observed includes: i)increased hemolysis; ii) increased K+leak; iii) reduction inpost-transfusion survival; and iv) reduced deformability. Such damage issimilar to, but an exaggerated form of, storage-induced damage of RBC.The compromised status of RBC is well known to the physicians whoadminister such compromised RBCs, and FDA mandates restricted use ofsuch RBCs in terms of shortened shelf life after gamma-irradiation (14days) and/or 28 days total shelf life for irradiated units.

The irradiation of blood components has received increased attention dueto increasing categories of patients eligible to receive such blood toprevent transfusion-associated graft versus host disease. However,irradiation also leads to enhancement of storage lesions, which couldhave deleterious effects when such blood is transfused. It is well knownin the field that the main deleterious side-effect of radiation on RBCis oxidative damage caused by ROS.

Radiation damage to RBC in the presence of oxygen can occur in two ways;

i) By ROS generated during and immediately after irradiation. ROS canattack proteins and lipids in vicinity, as well as to initiateperoxidation cycle of lipid and protein using oxygen to fuel.

ii) Met-Hb and its denaturation products generated in i) above act ascatalysts to further cause ROS-mediated oxidative damage duringsubsequent refrigerated storage of RBC. This is an enhanced version ofstorage lesion development using O₂.

ROS is a major culprit in causing deterioration of RBCs duringrefrigerated storage at blood banks Storing RBCs under anaerobicconditions significantly reduces such damages caused by ROS.Accordingly, deleterious or negative effects of gamma- and X-rayirradiation steps to RBCs, are substantially offset by the protectivebenefits oxygen and/or carbon dioxide removal and the subsequentanaerobic storage. Therefore, irradiation of RBCs is preferablyperformed after oxygen removal in OCDD 100 of FIG. 2. Additionally,gamma irradiation and X-ray irradiation can occur when RBCs are storedin storage bag 200 or subsequent to storage prior to oxygen additionbefore transfusion.

While oxygen, carbon dioxide or oxygen and carbon dioxide removal takeplace before irradiation, if irradiation occurs before oxygen removal(FIG. 1), oxygen removal should take place within twenty four hoursthereafter to limit effects of on RBCs.

Pathos n Inactivation

After RBCs have been collected, treatment for the removal of infectiousagents that may be present in donor blood and potentially passed torecipient 50 receiving transfusions, may be effected. Infectious agentsinclude microorganisms such as viruses, especially retroviruses,bacteria, fungi and non-microbial agents such as self-replicatingproteins (prions) and nucleic acids. A process called pathogeninactivation or reduction removes such dangerous infectious agents fromthe RBCs. However, the chemical processes of pathogen inactivation orreduction are also potentially damaging to the RBCs.

A pathogen inactivation process may involve chemical and light orriboflavin and light therapy. In an aspect, pathogen inactivation orreduction may be performed before storage in the anaerobic storageenvironment for whole blood, and for RBCs that have been separated fromwhole blood, before passage through OCDD or after passage through OCDDand before storage.

Since RBCs are stored in an anaerobic environment, growth of aerobicbacteria and parasites are prevented. Bacteria such as Yersiniaenterocolotica, Serratia liquefaciencs and Staphylococcus strains areanaerobic and require oxygen for growth and multiplication. Parasitessuch as Plasmodium falciparum (malaria protozoan), babsea (babesiosis)and Trypanosoma cruzi (Chagas disease) are documented infections in theUS following blood donations. These microaeophilic organisms and areexposed to varying oxygen concentrations during their life cycle. Theysurvive well in reduced oxygen environments, but may not adapt to theanaerobic conditions developed during RBC storage.

Due to the reduction of ROS production under anaerobic state, gamma orX-ray may be used at increased dosage and/or time compared to T cellinactivation for pathogen inactivation.

Blood Storage Bag

Referring to FIGS. 2 and 11 a, a blood storage bag 200 according to anembodiment of the present disclosure is provided. Blood bag 200 has aninner blood-compatible bag 250 (preferably polyvinyl chloride (PVC)),and an outer barrier film bag 255. The material of bag 250 is compatiblewith RBCs. Disposed between inner bag 250 and outer oxygen barrier filmbag 255 is a pocket that contains a sorbent 110. Barrier film bag 255 islaminated to the entire surface of inner bag 250. Sorbent 110 iscontained in a sachet 260, which is alternately referred to as acontainer, enclosure, envelope, pouch, pocket, etc. Sorbent 110 isoptimally located between tubing 440 that leads into and from bag 200and port 415, and specifically between inner bag and outer oxygenbarrier film bag 255. This location will ensure that oxygen disposedbetween these two bags will be scavenged or absorbed. Sorbent is ideallylocated in a sachet 260 and not in contact with RBCs. Sorbent mayinclude oxygen sorbents, and may also be combined with carbon dioxidesorbents, enabling sorbent 110 to deplete both oxygen and carbon dioxideat the same time.

Referring to FIG. 11b , storage bag 202, is similar to bag 200 ;however, bag 202 is a laminated bag. Bag 202 has a inner PVC blood bag210, an outer barrier bag 216, and a sorbent layer 215 between blood bag210 and outer barrier bag 216.

In FIG. 12A, a small sachet 210 contains sorbent 110. Small sachet 210is enclosed inside of PVC bag 205 and is preferably made from a siliconeor siloxane material with high oxygen permeability of biocompatiblematerial. Sachet 210 has a wall thickness of less than 0.13 mm thicknessensures that O₂ permeability ceases to become the rate-limiting step.PVC bag 205 may also contain carbon dioxide sorbent. Again, sorbent 110may be an oxygen, carbon dioxide and oxygen and carbon dioxide sorbent.

In FIG. 12B, RBCs are stored in storage bag 307 that has a secondary bag301 in order to maintain an anaerobic storage environment for RBCstorage. Secondary bag 301 may have a transparent oxygen barrier films(e.g., nylon polymer) that compensate for the inability of PVC blood bag305 operate as a sufficient oxygen barrier to maintain RBCs in ananaerobic state. Secondary bag 301 may be made with an oxygen barrierfilm, preferably a nylon polymer or other transparent, flexible filmwith low oxygen permeability. Bag 307 contains a sorbent sachet 310containing a sorbent 110 that is an oxygen, carbon dioxide or oxygen andcarbon dioxide sorbent.

Referring to FIGS. 12A and 12B, blood storage bags 201 and 301 areconfigured to store RBCs for extended storage periods of time in ananaerobic storage environment. Inner blood storage bags 205 and 305 arepreferably made from DEHP-plasticized PVC and are in contact with RBCs.DEHP-plasticized PVC is approximately 200 fold less permeable to oxygencompared to silicone. Inner storage bags can also be made from non DEHPplasticized PVC or other non DEHP plasticized polymer. DEHP has aprotective effect on the RBC membrane, but this effect is unnecessarywhen the RBCs are stored anaerobically.

However, PVC is insufficient as an oxygen barrier to maintain theanaerobic state of RBCs throughout the storage duration. Therefore,blood storage bags 201 and 301 may be fabricated with outer transparentoxygen barrier film 206, 306 (e.g. nylon polymer, aluminum oxide coatednylon etc.) laminated to the outer surface inner blood bag 205 and 305.This approach, as well as one shown in FIG. 1, uses accepted plasticmaterials for blood contact surface (for case of DEHP/PVC, supplyingDEHP for cell stabilization) at the same time prevents oxygen entry intothe bag during extended storage.

Alternatively, transparent organic oxygen sorbent film may be laminatedbetween 205/206 or 305/306 in place of 210/110 or 310.

OCDD 101 and various storage bags of the present disclosure can be usedin varying combinations. For example, OCDD 101 of FIG. 1 can be usedwith blood bag of FIGS. 11, 11 b, 12A or 12B. Other combinations andconfigurations are fully within the scope of the present disclosure.

During storage in bag 200 different components may be added to RBCsstored anaerobically and during carbon dioxide depletion. In addition toadditives, metabolic supplements may also be added to red blood cells.Metabolic supplements can be provided to RBCs specified times and ratesor frequencies by a metering device placed within the main storage bag,or added through pre-connected PVC bags. Metabolic supplements are addedto RBCs during storage at 4° C. Red blood cell storage extends wellbeyond the current 6-week limit for 12 or up to 20 weeks at 4° C., withlevels of 2-3 DPG and ATP that are above those found in freshly drawnblood. The metabolic supplement includes pyruvate, inosine, adenine, andoptionally dibasic sodium phosphate and/or monobasic sodium phosphate.Additionally, nutrient supplementation may optionally includesupplements that provide antioxidants to the storage medium, including,but not limited to analogues of reduced glutathione, vitamin C andvitamin E. Current refrigeration storage technology is essentially apremature aging process of RBCs in contrast to the metabolism protectionsystem of the present disclosure. Current refrigerated storage of redblood cells does not maintain appropriate cellular glutathionc levels.Glutathione supplementation may extend the storage time of RBCs. Theamounts and timing of glutathione supplementation may conveniently bedetermined and optimized as necessary.

Referring to the drawings and in particular to FIG. 13, anotherembodiment of a disposable blood anaerobic storage system is shown andreferenced using reference numeral 1000. The blood storage systemincludes a blood collection bag 1010, a combination oxygen/carbondioxide depletion device 1060 that includes a leukoreduction filter 1064and a oxygen and/or carbon dioxide depletion portion 1066, and ananaerobic blood storage bag 1070. System 1000 also includes a collectionbag 1030 for liquid plasma and/or platelets. Combination OCDD 1060 notonly removes oxygen and carbon dioxide from red blood cells travelingtherethrough, but also filters excessive white blood cells from the redblood cells. This embodiment of FIG. 14 offers a single-use, disposable,low cost system. Tubing connects the various components of the bloodstorage system 1000. Tube 1042 connection bag 1010 connects collectionbag 1010 and bag 1030 and tubing 1044 connects bag 1030 and bag 1040.Tube 1055 cnnects combination OCDD 580 with additive bag 1040.

In certain embodiments of the present disclosure, the system recognizesthat blood cells in storage continue to metabolize. It is desirable toreduce their metabolic rate as low as possible over time of storage, andyet maintain healthy viable cells that are of high quality fortransfusion. In an embodiment, the present disclosure uniquely protectsessential metabolism, prolongs the shelf life of refrigeratederythrocytes, and provides high quality blood product. Further,refrigeration reversibly disables the enzymes essential for met-Hbreduction in vivo, increases the solubility of damaging O₂ (almost by afactor of 2) in the environment of the red blood cells, and permits thelevel of ATP to decrease by diminishing the glycolytic rate (at 4° C.the rate is about 1% of that found at 37° C.). Reduction of red cell ATPconcentration results in echinocyte (i.e. an unstable form of red bloodcells) formation, increased rates of membrane vesiculation, loss of redcell surface area, and accelerated sequestration by splenic macrophages.Vesiculation continues throughout the cold storage period, isexacerbated by echinocyte formation, and decreases red blood cellsurvival by decreasing red blood cell membrane area.

Oxygen removal can be conducted at any temperature that maintains goodviability of the RBC. Preferably, oxygen is removed between about 1° C.and about 37° C. provided that RBC viability is maintained. Once in anembodiment of a blood storage device of the present disclosure, the RBCcan be stored under refrigeration in the manner consistent with commonindustry practice for storage of blood products, preferably at atemperature between 1° C. and 10° C., and more preferably at about 4° C.Such storage periods range from about 6 to about 20 weeks and longer.Preferred storage periods are about 6 to about 15 weeks duration orlonger provided RBC quality is maintained.

Pre-Transfusion

Prior to transfusion of stored RBCs to a patient or recipient, variousprocesses can be affected to maximize acceptance of RBCs by therecipient and to optimize the condition of the RBCs.

In those patients who are either small or whose circulatory systemscannot process a great influx of RBCs, the volume of the RBCs must bereduced immediately prior to transfusion. Such patient who may face suchan issue include those suffering from congestive heart failure orneonates. Volume reduction can be accomplished using a variety ofmethods.

When RBCs are stored for a length of time, the RBCs may generally bestored in a storage bag, such as bags of FIGS. 11 a, 11 b, 12A, and 12B.In some aspects, storage bags can have a hydrophilic membranecompartment in the top ½ of the bag, such as that of bag 208 of FIG. 14.In an aspect, bag 208 may have a hydrophilic membrane 207 having amembrane pore size must be less than {cube root}4 micron to retain theRBCs cells and to prevent them from flowing through. When membrane 207filled with a concentration of RBC with low hematocrit, plasma andadditive solution will pass through the membrane into the lowercompartment 206 concentrating RBCs in membrane 207. The top portion ofthe lower compartment needs a check valve 209 so the fluid will notescape during transfusion. Bag 208 may have a sorbent 101, as discussedabove, for purposes of continued depletion of oxygen, carbon dioxide,and oxygen and/or carbon dioxide.

More conventionally, a portion of RBC of low hematocrit can flow into asmall hollow fiber/film device having hydrophilic fibers/films, such asthe fibers/films of OCCD 100. A portion of the RBC will flow into thefiber/film lumens and liquid and the liquid portion will pass throughthe fiber/film wall. A differential pressure across the fiber/film wallwill be used to control RBC and fluid flow. This is another method ofconcentrating the RBCs in advance of transfusion.

Alternatively, RBCs may be concentrated by passage through severalmicrofluidic chips that use the inertia of the RBCs. The microfliudicchips harness the inertia of the RBCs by forcing the RBCs to flowthrough a plurality of narrow channels such that only one cell is ableto pass through each of the plurality of channels at a time. The cellsare in the center of the channel, they exit through a center outletport, and fluid, plasma and additives can exit in ports adjacent to thecenter port. The microfluidic chip may be scaled up for volumes.Microfluidic chips may contain at least one network unit disposed in asubstrate. Microfluidic devices have an aspiration pressure to enablesmovement of RBCs through the network unit.

A further processing step that is necessary immediately prior totransfusion is the introduction of nitric oxide (NO) to the RBCs toenhance vasoregulatory function. There is increasing awareness thatblood transfusion using banked blood is not only providing fullyperceived benefits, but in some cases, harmful to some recipients. Oneof the major reasons behind lower-than-expected efficacy of transfusedblood is postulated to be the loss of vasoregulatory function of RBCcaused by degradation of nitric oxide (NO) sequestered in hemoglobin(Hb) molecules within RBC. A recent report showed that as short as 3hours after blood collection, NO in RBC was lost, and its vasoregulatoryactivity can be restored with addition of NO replenishing compounds.Accordingly, the introduction of NO to RBCs during storage in blood bag200, immediately prior to transfusion and after storage will assist therecipient in receiving optimal benefits from the transfusion. Because ofincreased stability of NO in anaerobic conditions, nitric oxide is addedto the anaerobic environment of storage bag 200 prior to transfusion,for example. Additionally, NO can be added in the post-storage phase Cprior to the addition of oxygen before transfusion. NO addition requiresprior oxygen removal due to its inherent instability in the presence ofoxygen. Additionally, NO must be added immediately before transfusion inthe form of NO gas, NO precursor reagents, or nitrite. NO can be addedto RBCs in storage bag 200 using a small bag or cartridge to injectabove materials in form of a gas or nitrate or other precursor chemicalas part of a transfusion set.

Immediately before transfusion, oxygen can be supplied to RBCs tooxygenate RBCs. Addition of oxygen must be accomplished duringpost-storage phase C after gamma and x-ray irradiation and nitric oxideaddition, preferably immediately before transfusion at the bedside. Thepresence of oxygen with the processes of gamma and X-ray irradiation andthe addition of nitric oxide are deleterious to the RBCs as discussedabove.

The benefits of oxygen removal and or carbon dioxide removal from RBCsbefore storage in combination with and other therapies has a positiveeffect on the outcome of the stored RBCs in advance of transfusion.

FIGS. 15, 16 and 17 show the benefits offered by the proposed storagesystem and process of the present disclosure. In Phase I, represented bydotted line, RBCs are flushed with an inert gas to remove oxygen and arestored in an anaerobic canister for 9 weeks. In Phase 2, represented bysolid line, RBCs are oxygen, carbon dioxide, or oxygen and carbondioxide depleted in OCDD 100 and are stored in anaerobic storage bag 200for 9 weeks. Phase 2 shows that ATP levels of RBCs are significantlyhigher at weeks 3 through 9. By maintaining high levels of ATP, RBCsmaintain the ability of dilate the pre-capillary arteriole maintain ahigh metabolic level. Further, ATP can be boosted and significantlystimulated during the first four weeks of storage by initially depletingoxygen levels and maintaining carbon dioxide levels in the presence of awide range of additives. FIG. 15 shows that the shelf-life of RBCs issubstantially enhanced by reducing oxidative damage at 1-6° C. andmaintaining high levels of ATP for an extended period.

FIG. 16 shows that under Phase 2 anaerobic conditions, 2,3 DPG ismaintained at a high level by depleting carbon dioxide at the onset ofstorage. The transfusion of high 2,3 DPG blood with full oxygen carryingcapacity comparable to fresh blood provides significant benefits topatients with critical and immediate oxygen needs. The rate at which2,3, DPG declined after week 3 is typical.

Referring to FIG. 17, hemolysis is significantly lower during Phase 2 ascompared to hemolysis during Phase 1. In particular, hemolysis issignificantly lower at weeks 6 through 9 of storage. Hemolysis is aconcern for all transfused patients and is particularly a concern forpatients under chronic transfusion therapy. Patients with inheritedhemoglobinopathies such as sickle cell disease (SCD), alpha- andbeta-thalassemia, require repeated periodic transfusions of 30 or moreunits per year. These patients' RBCs have defective hemoglobin that doesnot function properly in gas transport, and often have RBCs of limitedlife span. These patients' own RBCs, together with RBCs from chronictransfusion therapy, can overload the body's capacity for iron.Long-term iron overload is highly toxic, and the complications thatarise from it become a main source of morbidity unless patients areplaced under continuous iron chelation therapy. One of the major sourcesof excess iron for chronically transfused patients is hemoglobinoriginating from non-viable RBCs (as a result of accumulated storagelesions) that are destroyed immediately after transfusion. By reducingthe number of non-viable RBCs, anaerobic storage of RBCs with higher24-hr recovery reduces addition of excess iron to these patients.

RBC storage life can be measured by the extent of vesicle formation,extent of hemolysis, and total cellular ATP levels. Long storage life isobtained when the tesicle formation is low, hemolysis is low and highATP levels are sustained, preferably above about 2-3 .mu.mol ATP per gHb. All of these parameters may be measured by the conventional methodsknown to those of skill in the art. For example, samples of cells can beassayed for the extent of hemolysis by calculating the fraction ofsupernatant hemoglobin relative to total hemoglobin. To measure ATPlevels, for example, RBCs can be assayed for ATP according to themethods described in Technical Bulletins 336-W and 35—(Sigma ChemicalCo., St. Louis, Mo.).

As used herein, improved or prolonged shelf life or improved storage ofRBCs refers to the preservation of viable RBCs for an extended period oftime relative to the current standard of about 6 weeks. In certainembodiments of the present disclosure, substantial oxygen removalprovides RBCs with an extended storage life of about 7-15 weeks. Inother embodiments according to the present disclosure, substantialoxygen removal provides RBCs with an extended storage life up to 20weeks or greater, particularly when cells are suspended in the storagesolutions provided by the present disclosure. In another aspect,substantial oxygen removal provides RBCs with an extended storage lifeof about 10-15 weeks. In other aspects, the extended storage life may be10 to 20 weeks or 10 to 25 weeks. In a further aspect, storage life canbe prolonged by preventing 2,3-DPG feedback inhibition of the RBCglycolytic pathway.

The in vitro parameters measured after storage of RBCs provide a meansto measure in vivo survival of RBCs. The conventional means to assess invivo survival is to determine the percentage of cell survival 24 hourspost transfusion in a recipient. Typically in the USA, the averagepercentage of cell survival needs to be about or better than 75% toprovide an acceptable RBC product. The three parameters, vesicleproduction, extent of hemolysis, and ATP levels, may be routinely usedindividually in the art to predict in vivo cell survival.

Referring to FIG. 18, immediately before RBCs are to be transfused, suchRBCs can be placed in a further bag 35 in connected to a device 107 toadd back oxygen prior to transfusion. Significantly, oxygen may be addedback to RBCs after any gamma or x-ray irradiation or addition of nitricoxide to avoid the development of deleterious storage legions addressedabove. Device 107 is like device OCDD, however, it does not have O₂/CO₂sorbent material, but instead contains pure oxygen or air in the innerspace containing hollow fibers. This use is for special cases, such asmassive transfusions, where the capacity of the lung to re-oxygenatetransfused blood may not be adequate, or sickle cell anemia. Once theoxygen is added back, transfusion using needle 405 can occur.

Referring to FIG. 19, a possible configuration of FIG. 1 shows oxygendepletion of RBCs and leukoreduction of RBCs prior to storage ananaerobic storage bag. FIG. 19 shows whole blood that may be obtainedfrom a donor or by apherisis, may be separated into components ofplasma, platelets and RBCs. An additive solution may be added to RBCsthat are either leukoreduced prior to oxygen, carbon dioxide or oxygenand carbon dioxide depletion or leukoreduced after oxygen, carbondioxide or oxygen and carbon dioxide depletion.

FIG. 20 illustrates a configuration of the flowchart of FIG. 1,including leukoreduction, oxygen, carbon dioxide or oxygen and/or carbondioxide depletion. FIG. 20 illustrates the step of leukoreduction duringthe whole blood or after component separation at which time RBCs can beleukoreduced. Additionally, leukoreduction can be conducted using acombination leukoreduction device 1060 that is also an oxygen and carbondioxide depletion device. Gamma irradiation or x-ray irradiation canoccur at various times during Phase A, Phase B or Phase C. Gamma andx-ray irradiation can occur during anaerobic conditions after theremoval of oxygen in OCDD device 100, during storage in anaerobicstorage bag 200 or in post-storage phase before the addition of oxygenprior to transfusion. Irradiation is preferably performed in ananaerobic environment because an anaerobic environment minimizesoxidative damage by removing fuel of oxidative reactions. Alternatively,when gamma or x-ray irradiation occur before anaerobic conditions arepresent, RBCs must undergo oxygen depletion shortly thereafter andpreferably within 24 hours.

FIG. 21 illustrates a configuration of the flowchart of FIG. 1,including leukoreduction, oxygen, carbon dioxide or oxygen and/or carbondioxide depletion and re-oxygenation immediately prior to transfusion toa recipient. Addition of oxygen immediately prior to transfusion isbeneficial to recipient of RBCs. Oxygen addition is particularlybeneficial to recipients of massive transfusions such as those whosuffer from sickle cell disease.

FIG. 22 illustrates a configuration of the flowchart of FIG. 1,including leukoreduction, oxygen, carbon dioxide or oxygen and/or carbondioxide depletion, and pathogen inactivation at various possible timesduring collection and storage. Pathogen inactivation may harm RBCs bygenerating reactive oxygen species during the process. Anaerobicenvironments reduce ROS RBC damage during subsequent storage period.

FIG. 23 illustrates a configuration of flowchart of FIG. 1, includingleukoreduction, oxygen, carbon dioxide or oxygen and/or carbon dioxidedepletion, and nitric oxide addition at various possible times duringstorage. Nitric oxide can be added as NO-precursors, NO gas or nitrateto anaerobic RBCs. The nitric oxide and hemoglobin-NO compound are lesslabile under anaerobic conditions.

In each of FIGS. 19 through 23, other processes described in FIG. 1 andthroughout present disclosure could also be provided to such figures.

Although the foregoing describes various embodiments by way ofillustration and example, the skilled artisan will appreciate thatvarious changes and modifications may be practiced within the spirit andscope of the present application.

1.-39. (canceled)
 40. A method of preparing red blood cells fortransfusion in the treatment of an inherited hemoglobinopathy in asubject in need thereof comprising: obtaining whole blood; separatingred blood cells from said whole blood to form packed red blood cells;depleting oxygen and carbon dioxide from said packed red blood cellsprior to storage to prepare depleted packed red blood cells; and storingsaid depleted packed red blood cells in an anaerobic storage bagcomprising an outer bag having a barrier film that is impermeable tooxygen and carbon dioxide, and an inner bag in contact with said redblood cells, wherein said depleted packed red blood cells are maintainedin an anaerobic condition, to form stored depleted red blood cells. 41.The method of claim 40, further comprising oxygenating said storeddepleted red blood cells.
 42. The method of claim 40, further comprisingtransfusing said red blood cells into a subject in need thereof.
 43. Themethod of claim 40, wherein said inherited hemoglobinopathy isthalassemia.
 44. The method of claim 40, wherein said inheritedhemoglobinopathy is alpha-thalassemia.
 45. The method of claim 40,wherein said inherited hemoglobinopathy is beta-thalassemia.
 46. Themethod of claim 40, wherein said stored depleted red blood cellscomprise an initial oxygen saturation of less than 3 Torr.
 47. Themethod of claim 40, wherein said subject in need thereof requires 30 ormore units of blood per year.
 48. The method of claim 40, wherein saidstored depleted red blood cells have higher 24-hr recovery and reducedexcess iron when transfused into said subject in need thereof, comparedto red blood cells not oxygen depleted.
 49. The method of claim 40,further comprising reducing iron overload in said subject in needthereof.
 50. The method of claim 40, wherein said stored oxygen depletedred blood cells are stored for up to 42 days.
 51. The method of claim40, wherein said anaerobic storage bag further comprises a sorbentcomprising an oxygen sorbent, a carbon dioxide sorbent, or a combinationthereof, disposed between said inner bag and said outer bag.
 52. Themethod of claim 40, further comprising adding an additive solution tosaid packed red blood cells to form a suspension of packed red bloodcells.
 53. The method of claim 51, wherein said additive solutioncomprises AS-1, AS-3, AS-5, SAGM, PAGG-SM, PAGG-GM, MAP, SOLX, ESOL,EAS61, OFAS1, OFAS3, or any combinations thereof.
 54. The method ofclaim 40, wherein said anaerobic storage bag is irradiated by gamma orx-ray irradiation.
 55. The method of claim 40, further comprising gammaor x-ray irradiating said depleted packed red blood cells beforetransfusion.
 56. The method of claim 40, wherein said red blood cellsreduce complications from chronic transfusion therapy in said subject inneed thereof.
 57. The method of claim 56, wherein said red blood cellshave higher 24-hr recovery and reduced excess iron when transfused intosaid subject in need thereof, compared to red blood cells not oxygendepleted.
 58. The method of claim 42, wherein said red blood cellsreduce morbidity from chronic transfusion therapy in a subject in needthereof.
 59. The method of claim 58, wherein said stored depleted redblood cells have higher 24-hr recovery and reduced excess iron whentransfused into said subject in need thereof, compared to red bloodcells not oxygen depleted.